Human cytomegalovirus (HCMV) causes substantial morbidity and mortality, primarily in newborns and immunocompromised patients. Like other viruses, HCMV must evade myriad host cell antiviral defense mechanisms, among which is the shut off of overall protein synthesis mediated by the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase R (PKR). HCMV encodes two proteins, pTRS1 and pIRS1, that block PKR activation. In addition to self-associating and to binding to dsRNA and to PKR, these proteins have the unprecedented effect of causing PKR to relocalize to the nucleus. In order to elucidate the mechanism and significance of pTRS1 and pIRS1 effects on PKR, experiments are proposed to precisely delineate the domains of pTRS1 and pIRS1 responsible for their interactions with RNA and proteins. These studies will also reveal whether the nuclear relocalization of PKR by pTRS1 and pIRS1 serves as an unusual viral strategy for removing PKR from its cytoplasmic targets or as a means of facilitating an as-yet-unidentified nuclear function of PKR that may be beneficial to the virus. Finally, experiments will directly test the hypothesis that either pTRS1 or pIRS1, and in particular their ability to bind to dsRNA and PKR, is essential for HCMV replication. Understanding the mechanism by which HCMV interferes with the PKR pathway will contribute new perspectives into biochemical interactions required for PKR inhibition in other systems as well as insights into the significance of the PKR nuclear redistribution activity which thus far seems to be a unique property of betaherpeviruses. The insights gained from studies of these unconventional HCMV dsRNA-binding proteins will also contribute to identifying new dsRNA-binding proteins in other systems and for analyzing their functions in counteracting PKR and other dsRNA-activated responses. Finally, since mutant viruses lacking the ability to block PKR activation have greatly reduced virulence in animal models, results of these studies may have applications in the development of new antiviral strategies and for the development of live attenuated vaccines.